Summary/Abstract Lung cancer is the leading cause of cancer-related deaths both worldwide and in the United States. It is also one of the most genetically complex diseases ? the two major subtypes of non-small cell lung cancer (NSCLC), which are lung squamous cell carcinoma and lung adenocarcinoma rank 2nd and 3rd for the most mutations per megabase of DNA in a study of 21 different tumor types. Efforts to map these mutations to genetic regions have demonstrated that mutations in the BRG1-Associated Factor (BAF) complex, also known as the mammalian SWI/SNF complex, are common. Inactivating mutations in BRG1, which encodes the ATPase and helicase of the BAF complex, are present in 8-20% of NSCLCs. BRG1 (also known as SMARCA4) is often co- mutated with KRAS, and there are no targeted therapies for this genetic subtype of lung cancer. The BAF complex has long been known to be the epigenetic antagonist of the EZH2-containing Polycomb Repressive Complex 2 (PRC2), but exactly how these complexes interact in the cancer setting is not well understood. Recently, we demonstrated that EZH2 inhibitors synergize well with Topoisomerase II (TopoII) inhibitors such as etoposide in BRG1 mutant NSCLCs. Our data suggest that a key role for BRG1 in lung cancer cells involves facilitating TopoII complex function. Furthermore, our data suggest that combination of EZH2 inhibition and TopoII inhibition uncovers the vulnerabilities of BRG1 mutant tumor cells to DNA repair defects. In order to understand the mechanistic basis of the interactions between EZH2, BRG1 and TopoII, we propose to use a panel of human and mouse isogenic models in which the only difference is the presence or absence of BRG1. In Aim 1 we will identify mechanisms of DNA repair defects that occur with BRG1 deficiency in isogenic murine and human lung cancer cell lines by assessing markers of double strand break repair mechanisms, non- homologous end joining (NHEJ) and homologous recombination (HR). We will also determine if defects in DNA repair cause the sensitivity of BRG1 mutant lung tumors to dual EZH2i/etoposide treatment. In Aim 2 we will use mouse models to examine the preclinical effects of combination of EZH2 inhibition and etoposide against BRG1-null lung tumors. Genetically engineered mice and patient derived xenograft (PDX) models will be used to determine if the combination of EZH2 and TopoII inhibition is superior to standard of care therapies in place for BRG1-mutant lung cancers. In Aim 3 we will characterize the immune microenvironment of BRG1-deficient tumors and we will assess the impact of EZH2 inhibition with etoposide treatment on tumor immune cells. The potential impact of these studies is large because BRG1 is one of the most commonly mutated genes in NSCLC and there is currently no targeted therapy for this genetic subtype. In addition, EZH2 inhibitors have moved to Phase I trials for other tumor types, and the combination of EZH2 inhibition and orally bioavailable etoposide (VP-16) could rapidly move to trials if these preclinical studies suggest efficacy.